Botulinum neurotoxin is a Clostridial neurotoxin that produces flaccid paralysis by irreversibly inhibiting the release of acetylcholine (ACh) from nerve terminals at the motor endplate. While this ACh-related phenomenon has been well documented and is generally considered its primary mechanism of action, numerous researchers have evidence that botulinum neurotoxins may also act by impairing or inhibiting vesicular release of other neurotransmitters and/or neuroactive substances. See, e.g., Ashton et al. (1988) 50 J.Neurochemistry 1808; McMahon et al. 267 J.Biol.Chem. 21388; Blasi et al. (1994) 88 J.Physiol. 235.
The profound specificity of action of botulinum neurotoxin has contributed to the fact that this neurotoxin has become widely employed for its therapeutic potential in the treatment of a variety of involuntary movement disorders (Borodic et al. (1991), 4 Opthalmology Clinics of North America 491-504; Hambelton, P. (1992), 239 J. Neurol. 16-20; Schantz et al. (1992), 50 Microbiol. Rev. 1:80-99; Valtorta et al. (1993), 27 Pharmacol. Res. 1:33-44). Intramuscular injection of nanogram quantities of purified botulinum neurotoxin is the treatment of choice for a number of clinical indications including: blepharospasm, spasmodic dysphonia, hemifacial spasm, and adult onset spasmodic torticollis. Botulinum neurotoxin therapy has primarily involved intramuscular injection of the serotype A, however, the F and B serotypes are currently also considered clinically relevant (Greene et al. (1993), Botulinum and Tetanus Neuroneurotoxins (ed. B. R. DasGupta, Plenum Press, New York pp. 651-654). These additional serotypes hold the promise of alternative therapies for patients that develop resistance to the A neurotoxin. Moreover, these neurotoxins may also possess intrinsic properties that result in significant therapeutic advantages.
Botulinum serotype A and B neurotoxins exhibit a number of functional, structural, and mechanistic similarities. Both produce chemical denervation at the neuromuscular junction that appears to occur through a three step process resulting in inhibition of normal neurotransmitter release (Schmitt et al. (1981) 317 Naunyn-Schmiedeberg's Arch. Pharmacol. 326-330; Simpson, L. L. (1981) 33 Pharmacol. Rev. 155-158). Both species have a molecular weight of approximately 150,000 daltons, and the active form of the neurotoxin exists as a dimeric molecule consisting of a light (approximately 50,000 DA molecular weight) and heavy (approximately 100,000 DA molecular weight) subunit linked by a disulfide bond (Simpson, L. L. (1981) 33 Pharmacol. Rev. 155-158; Tse et al. (1982) 122 Eur. J. Biochem. 493-500).
Despite the general similarities between the A and B neurotoxins, however, closer examination of these species reveals very significant differences. For example, all of the neurotoxins produced by C. botulinum are immunologically distinct which suggests significant differences in their amino acid sequences. Analysis of the partial amino acid sequences for the A and B serotypes has revealed greater homologies between the primary and secondary structure for the light subunit chains than the heavy subunit chains. The degree of primary structure homology is only 20% for the light subunit chains versus 40% for the heavy (Evans et al. (1986) Eur. J. Biochem. 409-416). Although similar in secondary and tertiary structure, differences in the conformation of the neurotoxins at or near the active site may be responsible for the differences in neurotoxicity (Dasgupta, B. R. (1990) 84 J. Physiol. (Paris) 220-228).
Electrophysiological studies have demonstrated that botulinum neurotoxins affect different steps in the neurotransmitter release process. Botulinum serotype B affects synchronization of quantal transmitter release whereas serotype A does not (Singh et al. (1989) 86 Mol. Cel. Biochem. 87-95). Similarly, differences exist with regard to reversibility of the inhibition of calcium-dependent release of neurotransmitter. Introduction of calcium into nerve terminals using a calcium ionophore produces the release of transmitter from synaptosomes poisoned by serotype A more readily than those poisoned by serotype B (Molgo et al. (1990) 84 J. Physiol. (Paris) 152-166). At the neuromuscular junction, aminopyridine was also more effective at reversal of inhibition produced by botulinum neurotoxin A (Ashton et al. (1991) 56 J. Neurochem. 827-835). Ashton et al. (1991) have demonstrated that microtubule-dissociating drugs were effective in blocking the inhibitory effects of serotype B on neurotransmitter release and ineffective against the serotype A neurotoxin.
The differences in the toxic and neurophysiological effects of the serotype A and B neurotoxins may be related to the putative existence of two distinct receptor (acceptor) sites for these species (Singh et al. (1989) 86 Mol. Cel. Biochem. 87-95). Examination of the effect of botulinum neurotoxin A on .sup.125 I-botulinum neurotoxin B binding to neuronal membranes showed a very weak interaction involving both the high and low affinity sites for toxin binding.
Pearce et al. (1995) in 33 Toxicon 217 and Pearce et al. (1994) in 128 Toxicol. App. Pharmacology 69 have recently reported that a regional chemodenervation assay, which does not rely on the conventional LD.sub.50 calibration standard, more accurately predicts the clinical potency of botulinum neurotoxins. As disclosed by Pearce et al., this assay is useful to quantify more accurately the denervating potential of injections of individual botulinum neurotoxins. On the one hand, such injections of individual botulinum neurotoxins have proven clinically useful in creating regional muscular denervation to treat segmental movement diseases in man. On the other hand, however, one of the limitations of such traditional clinical therapy is that the individual neurotoxin must be repetitively injected into muscles every 10-14 weeks to effectively treat these chronic movement diseases. Some researchers have sought alternatives such as more potent preparations of neurotoxins which have demonstrated some clinical efficacy in animals. In contrast, other alternatives using conventionally calibrated doses of combinations of serotypes A and B have failed (In Therapy with Botulinum Toxin (1994) (ed., J. Jankovic and M. Hallett; Marcel Dekker, Inc., N.Y.)pp. vii-ix).
Clinically speaking, what is needed is a composition and a method which (a) act by increasing the duration of action of the chemodenervating agent so that repetitive injections can be given less often than the traditional clinical protocol currently prescribes; (b) are effective at dosage amounts which are less likely to stimulate a patient's immunogenic responses; and (c) accomplish the aforementioned clinical objectives with no or minimal adverse diffusion-dependent side-effects typically observed with prior art compositions and methods.
It is an object of this invention to provide compositions and methods which unexpectedly increase the beneficial effects derived from botulinum-based chemodenervation. It is a further object of the present invention to provide compositions and methods which utilize certain admixtures of neurotoxins to achieve potentiation of the temporal duration of chemodenervation which has heretofore been undescribed. It is another object of the present invention to provide certain compositions and methods which further permit more localized chemodenervation using admixtures of neurotoxins which contain lesser amounts of individual neurotoxins than heretofore has been described. It is yet another object of this invention to provide compositions and methods which further permit more localized chemodenervation using admixtures of neurotoxins which produce fewer diffusion-dependent side effects.